H2E-derived Ealpha52-68 peptide presented by H2Ab interferes with clonal deletion of autoreactive T cells in autoimmune thyroiditis

Abstract

Susceptibility and resistance to experimental autoimmune thyroiditis is encoded by MHC H2A genes. We reported that traditionally resistant B10 (H2(b)) mice permit thyroiditis induction with mouse thyroglobulin (mTg) after depleting regulatory T cells (Tregs), supporting A(b) presentation to thyroiditogenic T cells. Yet, Ea(k) transgenic mice, expressing A(b) and normally absent E(b) molecules (E(+)B10 mice), are susceptible to thyroiditis induction without Treg depletion. To explore the effect of E(b) expression on mTg presentation by A(b), seven putative A(b)-binding, 15-16-mer peptides were synthesized. Five were immunogenic for both B10 and E(+)B10 mice. The effect of E(b) expression was tested by competition with an Ealpha52-68 peptide, because Ealpha52-68 occupies approximately 15% of A(b) molecules in E(+)B10 mice, binding with high affinity. Ealpha52-68 competitively reduced the proliferative response to mTg, mTg1677, and mTg2342 of lymph node cells primed to each Ag. Moreover, mTg1677 induced mild thyroiditis in Treg-depleted B10 mice, and in E(+)B10 mice without the need for Treg depletion. Ealpha52-68 competition with mTg-derived peptides may impede clonal deletion of pathogenic, mTg-specific T cells in the thymus.

FIGURE 1

Five mTg-derived peptides are immunogenic in A⁺E⁻ and A⁺E⁺ mice. A, A⁺E⁻ or A⁺E⁺ mice were depleted of Tregs by injection of 1 mg CD25 mAb 14 and 10 days before immunization with mTg-CFA. LNC (6 × 10⁵ cells plus 4 × 10⁵ irradiated SC/well) were harvested at day 10 and cultured with indicated Ags for 5 days before proliferation was assessed by [³H]thymidine incorporation (background cpm 590–1400 ± 150–850). B, A⁺E⁻ and A⁺E⁺ mice were immunized with the indicated peptide in CFA and lymph nodes were removed on day 10. LNC were cultured with mTg-derived peptides (10 μg/ml) for 5 days and proliferation was determined by [³H]thymidine incorporation (background cpm 1100–3300 ± 30–340).

FIGURE 2

The five immunogenic mTg-derived peptides are H2A^b-resticted. A⁺E⁺ mice were immunized with the indicated peptide in CFA and lymph nodes were removed on day 10. LNC (6 × 10⁵ cells plus 4 × 10⁵ irradiated SC/well) were cultured with the immunizing peptide (10 μg/ml) in the presence of anti-A^b (Y-3P) or anti-E^b (Y-17) mAb for 5 days before proliferation was assessed (background cpm 1500–6300 ± 250–950). *, p < 0.05; **, p < 0.001.

FIGURE 3

Detection of the Eα52-68/A^b complex in H2E-expressing A⁺E⁺ mice. A, B220-gated APC from A⁺E⁻ (left) or A⁺E⁺ (right) SC were FACS analyzed for expression of Eα52-68/A^b (mAb Y-Ae, thick line) or total A^b (mAb Y-3P, dotted line) as a control. The dashed line shows background fluorescence. B, SC from A⁺E⁻ mice were incubated with Eα52-68 (thick line) or medium (dotted line) for 1 h and then labeled with mAb Y-Ae. The cells were FACS analyzed as in A.

FIGURE 4

Eα52-68 competes with native mTg as well as mTg1677 and mTg2342, but not mTg1744, for presentation by H2A^b. A, A⁺E⁻ mice were depleted of Tregs by injection of 1 mg CD25 mAb 14 and 10 days before immunization with mTg-CFA. LNC (6 × 10⁵ cells plus 4 × 10⁵ irradiated SC/well) were harvested at day 10 and cultured with mTg (40 μg/ml). Eα52-68 (10 μg/ml) was added to some wells to compete for presentation by A^b, and anti-A^b mAb (Y-3P) was added as a control to block presentation. The cells were cultured for 5 days before proliferation was assessed by [³H]thymidine incorporation (background cpm 1400 ± 850). B–D, A⁺E⁻ mice were immunized with the indicated peptide in CFA and lymph nodes were removed between days 10 and 12. LNC were cultured with the immunizing peptide (10 μg/ml), and Eα52-68 (10 μg/ml) and Y-3P were added as in A. Proliferation was determined by [³H]thymidine incorporation (background cpm 1400–3100 ± 850–1000). *, p < 0.05; **, p < 0.01.

FIGURE 5

Eα52-68 blocks presentation of mTg1677, but not mTg1744; neither can interfere with binding of Eα52-68 to A^b molecules. A and B, A⁺E⁻ mice were immunized with the indicated peptide in CFA and lymph nodes were removed on day 10. On day of culture, irradiated SC were incubated for 1 h with or without Eα52-68 (1° incubation, 10 μg/ml) and washed. The SC were subsequently incubated for another hour with the immunizing peptide (2° incubation, 10 μg/ml) and washed. These pulsed APC were then added to cultures (4 × 10⁵/well) along with peptide-primed LNC from immunized mice (6 × 10⁵/well) for 5 days. Proliferation was assessed by [³H]thymidine incorporation (background cpm: A, 1200 ± 160; B, 6800 ± 820). *, p < 0.01. C, A⁺E⁻ SC were incubated for 1 h with either mTg1677 or mTg1744 (10 μg/ml), or control medium, and washed. The SC were then incubated for 1 h with Eα52-68 (10 μg/ml), washed, and labeled with anti-B220 and anti-Eα52-68/A^b (mAb Y-Ae). B220-gated APC were analyzed by FACS for Eα52-68/A^b expression. The dotted line represents unlabeled APC, the filled histogram represents cells incubated with Eα52-68 only, the solid line represents cells incubated first with mTg1677 and then Eα52-68, and the dashed line represents cells incubated first with mTg1744 and then Eα52-68.

FIGURE 6

The immune response to mTg1677 is influenced by Tregs. A, A⁺E⁻ or A⁺E⁺ mice were depleted of Tregs by injection of 1 mg CD25 mAb 14 and 10 days before immunization; control mice were injected with 1 mg rat IgG. On day 0, mice were immunized with mTg1677-CFA, and on day 10 mice were killed. LNC (6 × 10⁵ cells plus 4 × 10⁵ irradiated SC/well) were cultured with mTg1677 for 5 days before proliferation was assessed by [³H]thymidine incorporation (background cpm 410–1600 ± 70–110). *, p < 0.05; **, p < 0.0001. B–D, Representative sections of thyroids from Treg-depleted, mTg1677-immunized mice (original magnification ×200 and ×400 for boxed inlays connected to areas by arrows). A⁺E⁻ or A⁺E⁺ mice were depleted of Tregs as described above and immunized with mTg1677-CFA on days 0 and 7. Thyroids were removed on day 35. B, Section from an A⁺E⁻ mouse without mononuclear cell infiltration. C, Section from an A⁺E⁻ mouse with destruction involving 15% of the thyroid. D, Section from an A⁺E⁺ mouse with destruction involving 30% of the thyroid.